摘要
目的 探讨人胚胎干细胞衍生的神经干细胞微囊泡(hESC-NSC-MVs)通过调节蛋白激酶B(AKT)/内皮型一氧化氮合酶(eNOS)信号通路促进坐骨神经损伤(SNI)大鼠坐骨神经再生和修复的作用.方法 取SD大鼠随机分为假手术组、模型组、hESC-NSC组、hESC-NSC-MVs组、hESC-NSC-MVs+CCT128930(AKT抑制剂)组,每组10只,模型组和实验干预组大鼠建立SNI模型,以hESC-NSC、hESC-NSC-MVs和CCT128930分组干预后,以坐骨神经功能指数(SFI)评测各组大鼠坐骨神经功能;检测各组大鼠坐骨神经传导速度、支配的腓肠肌恢复情况(以腓肠肌湿重比、肌纤维横截面积评测);透射电子显微镜(TEM)检测各组大鼠坐骨神经超微结构;试剂盒测定各组大鼠血清与坐骨神经eNOS和一氧化氮(NO)水平;免疫印迹实验检测各组大鼠坐骨神经AKT/eNOS信号通路相关蛋白表达.结果 假手术组相比,模型组大鼠坐骨神经超微结构发生显著损伤,SFI、坐骨神经传导速度、腓肠肌湿重比、肌纤维横截面积、eNOS和NO水平、p-AKT/AKT与eNOS蛋白表达显著降低(P＜0.05);与模型组相比,hESC-NSC组、hESC-NSC-MVs组大鼠坐骨神经超微结构损伤均减轻,SFI、坐骨神经传导速度、腓肠肌湿重比、肌纤维横截面积、eNOS和NO水平、p-AKT/AKT与eNOS蛋白表达均升高,且hESC-NSC-MVs对SNI大鼠上述各指标的作用更强(P＜0.05);与hESC-NSC-MVs组相比,hESC-NSC-MVs+CCT128930组大鼠坐骨神经超微结构损伤加重,SFI、坐骨神经传导速度、腓肠肌湿重比、肌纤维横截面积、eNOS和NO水平、p-AKT/AKT与eNOS蛋白表达降低(P＜0.05).结论 hESC-NSC-MVs可通过激活AKT/eNOS信号通路而减轻SNI大鼠坐骨神经超微结构及功能损伤,促使坐骨神经再生和修复,并改善其坐骨神经功能.
Abstract
Objective To investigate the role of human embryonic stem cell-derived neural stem cell microvesicles(hESC-NSC-MVs)in promoting sciatic neuroregeneration and repair in sciatic nerve injury(SNI)rats by regulating the protein kinase B(AKT)/endothelial nitric oxide synthase(eNOS)signaling pathway.Methods SD rats were randomly separated into sham surgery group,model group,hESC-NSC group,hESC-NSC MVs group,hESC-NSC MVs+CCT128930(AKT inhibitor)group,with 10 rats in each group.SNI models were established in the model group and experimental intervention group.After grouping and intervention with hESC-NSC,hESC-NSC-MVs,and CCT128930,the sciatic nerve function index(SFI)was applied to evaluate the sciatic nerve func-tion of each group.The conduction velocity of the sciatic nerve and the recovery of the innervated gastrocnemius muscle of rats in each group were measured using the wet weight ratio of the gastrocnemius muscle and the cross-sectional area of muscle fibers.Transmission electron microscopy(TEM)was applied to detect the ultrastructure of the sciatic nerve of rats in each group.Reagent kit was applied to measure the levels of eNOS and nitric oxide(NO)in the serum and sciatic nerve of rats in each group.Immunoblotting experiments were applied to detect the expression of AKT/eNOS signaling pathway related proteins in the sciatic nerve of rats in each group.Results Compared with the sham surgery group,the sciatic nerve ultrastructure of rats in the model group was obviously damaged,the SFI,sciatic nerve conduction velocity,wet to weight ratio of gastrocnemius muscle,muscle fiber cross-sectional area,eNOS and NO levels,and p-AKT/AKT and eNOS protein expression were obviously reduced(P＜0.05).Compared with the model group,the ultrastructural dam-age to the sciatic nerve in rats in the hESC-NSC group and hESC-NSC-MVs group was reduced,the SFI,sciatic nerve conduction ve-locity,wet to weight ratio of gastrocnemius muscle,muscle fiber cross-sectional area,eNOS and NO levels,and p-AKT/AKT and eNOS protein expression all increased,and hESC-NSC-MVs had a stronger effect on the above indicators in SNI rats(P＜0.05).Compared with the hESC-NSC-MVs group,the rat sciatic nerve ultrastructural damage was aggravated in the hESC-NSC-MVs+CCT128930 group,the SFI,sciatic nerve conduction velocity,wet to weight ratio of gastrocnemius muscle,muscle fiber cross-sectional area,eNOS and NO levels,and p-AKT/AKT and eNOS protein expression were reduced(P＜0.05).Conclusion HESC-NSC-MVs can alleviate the ultrastructural and functional damage of the sciatic nerve in SNI rats by activating the AKT/eNOS signaling pathway,promote sciatic neuroregeneration and repair,and improve its sciatic nerve function.
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